The abnormalities of gene regulation and protein synthesis associated with cancer result in reappearance of fetal antigens and isozymes, as well as disruption of normal cell differentiation. Cell surface changes may result in the appearance of tumor-associated or differentiation-associated antigens, as well as architectural rearrangements to produce subtle associative antigens. The high resolving power of monoclonal antibodies was used to analyze cell surface antigens associated with colon cancer cell lines LS180, LS174T and clones of LS174T. The LS174T/LS180 cell system possesses several properties associated with some colon cancers, namely, high CEA release, hypodiploidy, growth in nude mice, copious mucin production, the ability to differentiate in appropriate environments and binding of absorbed sera from some untreated colon cancer patients. Analysis of some biological properties of LS174T cell clones revealed that they differ 6.3-fold in CEA release into culture media after 21 days of incubation (p less than 0.01). The clones differ 3-fold in growth rates and in histological differentiation levels. BALB/c mice were immunized intraperitoneally (i.p.) with 3 x 107 trypsinized clone 3-5 cells and boosted i.p. on day 7 with 1 x 107 trypsinized and on days 14 and 18 with 1 x 107 cells scraped from culture flasks. The spleen cells harvested on day 19 were fused with a nonsecretor clone of P3 mouse myeloma cells at a 5:1 ratio using polyethylene glycol 1000. Hybridomas were evaluated for antibody using an indirect radioimmunoassay. Antibody binding to freshly trypsinized human tumor and normal cell lines was used to screen for specificity. The antigen recognized by MAB 5E113 can be extracted by 3M KCl, but not by 2% 1-butanol, suggesting that the antigenic epitope is borne on an integral membrane structure. Preliminary data suggest that the antigenic extracted by 3M KCl is between 5,000 and 30,000 MW. Studies are in progress to determine the chemical properties of this antigen and its relation to colon cancer cells. Future studies include immunization of mice with clone 3-5 cells growing in a differentiated state. Monoclonal antibodies will be screened for binding to cells growing as organoid glands on collagen gels. This work may establish the role of tumor-specific, as well as differentiation-associated, antigens in colon cancer.